Type 1 allergies have worldwide importance. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma.
These allergies are caused by sources of various origin, such as trees and grasses (pollen), fungi (spores), mites (excrement), cats or dogs. The allergen sources are released directly into the air (pollen, spores) or can reach the air bonded to diesel soot particles (pollen) or house dust (mite excrement, skin particles, hair). Since the allergy-triggering substances are located in the air, the term aeroallergens is also used.
The type 1 allergy-triggering substances are proteins, glycoproteins or poly-peptides. After uptake via mucous membranes, these allergens react with the IgE molecules bound to the surface of mast cells in sensitised persons. If these IgE molecules are crosslinked with one another by an allergen, this results in the secretion of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding allergic symptoms.
Up to 40% of type 1 allergy sufferers exhibit specific IgE reactivity with pollen extracts of true grasses (Burney et al., 1997, J. Allergy Clin. Immunol. 99:314-322; D'Amato et al., 1998, Allergy 53: 567-578; Freidhoff et al., 1986, J. Allergy Clin Immunology, 78, 1190-2002). The family of the true grasses (Poaceae) encompasses more than 10000 species, many more than 20 of which are hitherto known as triggers of allergic symptoms (Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107; Esch, 2008, Allergens and Allergen Immunotherapy, Clinical Allergy and Immunology Series, 107-126).
Most of the allergy-triggering true grasses belong to the Pooideae sub-family. Besides the grass species occurring as wild forms, such as, for example, Holcus lanatus (velvet grass), Phalaris aquatica (canary grass), Anthoxanthum odoratum (sweet vernal grass), Dactylis glomerata (orchard grass), Festuca pratensis (meadow fescue), Poa pratensis (Kentucky blue grass) or Lolium perenne (rye grass), cultivated cereals, such as Triticum aestivum (wheat), Secale cereale (rye) and Hordeum vulgare (barley), are also known members of this sub-family.
One of the Pooideae species which has been investigated best with respect to its allergens is Timothy grass (Phleum pratense), which is widespread worldwide as a wild plant and also plays a commercial role as a pasture plant and hardy feed grass.
Depending on the relative frequency in a population with which the individual allergen molecules react with the IgE antibodies of allergy sufferers, a distinction is made between major and minor allergens.
Six allergens of Timothy grass can be regarded as major allergens: Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthie-sen and Löwenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al., 1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54), Phl p 2/3 (Dolecek et al., 1993, FEBS 335 (3): 299-304), Phl p 4 (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int. Arch. Allergy Immunol. 97: 287-294; Nandy et al., Biochem. Biophys. Res. Commun., 2005, 337(2): 563-70) and Phl p 13 (Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402).
The dominant major allergens of Timothy grass are Phl p 1 and Phl p 5 (Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107), where Phl p 5 occurs in the two forms 5a and 5b, which are encoded by independent genes and differ with respect to their molecular weight. Following the official nomenclature of allergens, Phl p 5a is called Phl p 5.01 and Phl p 5b is called Phl p 5.02 (WHO/IUIS Allergen Nomenclature Subcommittee, www.allergen.org). The amino acid sequences both of Phl p 5a and also of Phl p 5b have been derived from the cloned cDNA sequences. Natural variants of both isoforms have been identified, which differ from one another through point mutations and correspond to different allelic forms (Vrtala et al., 1993, J. Immunol., 151: 4773-4781; Gelhar et al., 1997, Eur. J. Biochem., 247: 217-23). These variants are recorded in the WHO/IUIS database as Phl p 5.01xx and Phl p 5.02xx.
Natural Phl p 5a (nPhl p 5a) is a protein of about 32 kDa and reacts with IgE antibodies of 85-90% of grass pollen allergy sufferers (Rossi et al., 2000, Allergy Int., 49: 93-97).
The pollen of the related true grass species from the Poaceae family and in particular the Pooideae sub-family, such as Lolium perenne or Poa pratensis, contain allergens which are homologous with Phl p 5 and together are known as group 5 allergens. The high structural homology of these group 5 allergens causes correspondingly high cross-reactivity of the molecules with IgE antibodies (Lorenz et al., 2009, Int. Arch. Immunol. 148:1-17). Finally, this cross-reactivity means that sensitisation by one grass species may be sufficient to trigger an allergic reaction by other related grasses.
The high cross-reactivity of group 5 allergens is ultimately based on a similar primary sequence of the homologous allergens. This is shown by an amino acid sequence comparison of group 5 allergens of selected Pooideae species (FIG. 1).
Besides the cross-reactivity of the group 5 allergens with one another, cross-reactivity of Phl p 5 with another major allergen of Timothy grass is also known (Løwenstein, 1978, Allergy 33: 30-41; Petersen et al., 1995, Int. Arch. Allergy Immunol. 108: 55-59; Blume et al., 2004, Proteomics 4: 1366-71). The polypeptide chain of the allergen Phl p 6 exhibits great similarity with the N-terminal half of the various Phl p 5 sequences (FIG. 1). It is thought that the allergens can be traced back to a common original gene. The similarity between the allergens of the two groups has the effect that some of the Phl p 5-reactive IgE antibodies also bind to Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108: 49-54; Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107).
The 3D structure of many allergens has been explained in the past by NMR spectroscopy or X-ray structural analysis and served, inter alia, as the basis for localisation of IgE-binding epitopes on the protein surface. In the case of group 5 allergens of grass pollen, it has hitherto not been possible to generate a model which encompasses the entire polypeptide chain (Rajashankar et al., 2002, Acta Cryst. D58:1175-1181; Maglio et al., 2002, Protein Engineering 15: 635-642).
On the basis of 3D structures of the allergen Phl p 6 (RCSB protein data bank entry: 1NLX) and of a Phl p 5b half-molecule (RCSB protein data bank entry: 1L3P), it has been possible to generate a homology model of Phl p 5a (Wald et al., 2007, Clin. Exp. Allergy 37:441-450). According to this model, Phl p 5a is built up from two helix bundles, but the precise position of the two bundles to one another cannot be explained by the homology model (FIG. 2).
Specific immunotherapy (SIT) or hyposensitisation is regarded as an effective approach to the therapeutic treatment of allergies (Fiebig 1995 Allergo J. 4 (6):336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562); Cox et al., 2007, J. Allergy Clin. Immunol. 120:S25-85; James & Durham, 2008, Clin. Exp. Allergy 38: 1074-1088).
The classical therapy form of injection therapy (SCIT), in which natural allergen extracts are injected subcutaneously into the patient in increasing doses, has been used successfully for about 100 years. In this therapy, the immune system of the allergy sufferer is repeatedly confronted with allergens, causing reprogramming of the immune system to be achieved together with tolerance of the allergens. After uptake of the antigens from the allergen preparations by antigen-presenting cells, peptides are presented to the antigens on the cell surface. Some particular peptides which contain so-called T-cell epitopes are recognised by antigen-specific T-cells. This binding results, inter alia, in the development of various types of T-cells having a regulatory function. In the course of SIT, the regulatory T-cell response results in tolerance of the allergen, the downregulation of TH2 cytokines, the restoration of the TH1/TH2 equilibrium, the suppression of allergen-specific IgE, the induction of IgG4, IgG1 and IgA antibodies, the suppression of effector cells (mast cells, basophils and eosinophils) and the renewal of inflamed tissue (Akdis et al., 2007, J. Allergy Clin. Immunol. 119 (4):780-789; Larchè et al., 2008, Nature Reviews 6:761-771). The T-cell epitopes are thus of crucial importance for the therapeutic action of allergen preparations in the case of hyposensitisation.
Owing to the cross-reactivity of the major allergens of the true grasses which is present at the IgE and also the T-cell level, successful therapy with an allergen extract of a single representative grass species is usually sufficient (Malling et al., 1993, EAACI Position Paper: Immunotherapy, Allergy 48: 9-35; Cox et al., 2007, J Allergy Clin Immunol 120: 25-85).
Besides subcutaneous immunotherapy, a sublingual therapy form, in which the allergens or allergen derivatives are taken up via the oral mucous membrane, is undergoing clinical trials and use as an alternative to injection therapy (James & Durham, 2008, Clin. Exp. Allergy 38: 1074-1088).
A further possibility is treatment with expressible DNA which encodes for the relevant allergens (immunotherapeutic vaccination). Experimental evidence of the allergen-specific influencing of the immune response has been furnished in rodents by injection of allergen-encoding DNA (Hsu et al. 1996, Nature Medicine 2 (5):540-544, Weiss et al., 2006, Int. Arch. Allergy Immunol. 139: 332-345).
In all these therapy forms, there is a fundamental risk of allergic reactions or even anaphylactic shock (Kleine-Tebbe, 2006, Allergologie, 4:135-156). In order to minimise these risks, innovative preparations in the form of allergoids are employed. These are chemically modified allergen extracts which have significantly reduced IgE reactivity, but identical T-cell reactivity compared with the untreated extract (Fiebig 1995 Allergo J. 4 (6):336-339, Kahlert et al., 1999, Int. Arch. Allergy Immunol, 120: 146-157).
Therapy optimisation is possible with allergens prepared by recombinant methods. Defined cocktails of high-purity allergens prepared by recombinant methods, which are optionally matched to the individual sensitisation patterns of the patients, could replace extracts from natural allergen sources, since, apart from the various allergens, the latter contain a relatively large number of immunogenic, but non-allergenic accompanying proteins. Initial clinical studies with recombinant allergens have already been carried out with success (Jutel et al., 2005, J. Allergy Clin. Immunol., 116: 608-613; Valenta & Niederberger, 2007, J. Allergy Clin. Immunol. 119: 826-830).
Realistic prospects which may result in safe hyposensitisation with recombinant expression products are offered specifically by mutated recombinant allergens in which IgE epitopes are modified without impairing the T-cell epitopes which are essential for the therapy (Schramm et al. 1999, J. Immunol. 162:2406-2414). These hypoallergenic proteins could be employed in relatively high doses during SIT without increasing the probability of undesired IgE-promoted side effects.
In the past, such “hypoallergenic” variants with reduced IgE binding have been published for many aeroallergens (inter alia pollen and house dust mite allergens) and food allergens. On the basis of the DNA of unmodified allergens, it has been possible to prepare and express a recombinant DNA, inter alia by fragmentation, oligomerisation, deletions, point mutations or recombination of individual sections of an allergen (DNA shuffling) (Ferreira et al., 2006, Inflamm. & Allergy—Drug Targets 5: 5-14; Bhalla & Singh, 2008, Trends in Biotechnology 26:153-161).
With respect to grass pollen allergens, hypoallergenic variants of groups 1, 2, 5a, 5b, 6, 7 and 12 have been described (Ferreira et al., 2006, Inflamm. & Allergy—Drug Targets 5: 5-14; Westritschnig et al., 2007, J. Immunol. 179: 7624-7634).
A number of publications have to date described approaches to the development of hypoallergenic group 5 allergens. For Phl p 5a and Phl p 5b, it has been shown that the combined deletion of two sequence sections results in a considerable reduction in IgE binding and reduced ability to stimulate basophilic effector cells. However, the T-cell reactivity of the deletion mutants was modified slightly compared with that of the unmodified allergen (Schramm et al., 1999, J. Immunol. 162: 2406-2414; Wald et al., 2007, Clin. Exp. Allergy 37:441-450).
In another paper, the group 5 allergen from Lolium perenne (Lol p 5) was modified by amino acid substitutions and/or short deletions at the C terminal (Swoboda et al., 2002, Eur. J. Immunol. 32: 270-280). The mutations were not restricted to a particular amino acid type. The substituted amino acids were lysine, phenylalanine, threonine, valine or alanine. The conceptional approach of generating hypoallergenic mutants by specific mutation of a number of residues of an individual amino acid has likewise been described. Gelhar et al. described the generation of a recombinant Phl p 5b fragment by substitution of ten lysine residues localised at the protein surface by alanine (Gelhar et al., 2006, Int. Arch. Allergy Immunol. 140:285-294). However, a mutation strategy based on point mutations in proline residues has hitherto not been published for group 5 grass pollen allergens.
The object on which the present invention is based consisted in the provision of novel recombinant variants of group 5 allergens of the Poaceae at the protein and DNA level which are distinguished by reduced IgE reactivity at the same time as substantial retention of the T-cell reactivity and are therefore suitable for curative and preventive specific immunotherapy and immunotherapeutic DNA vaccination.